The use of sensors with an electroconductive polymer film as a sensing element is one of the most promising and attractive methods of quantitative electrochemical analysis [1]. To date, a number of electrochemical sensors based on electroconductive polymers have been described. They are distinguished by the principle of the measurement (amperometric, voltamperometric, chemoresistive, potentiometric) as well as by the method of receiving the analytical signal (direct and non-direct sensors).
The amperometric signal is received by applying to a sensor a constant voltage from external source and measuring a level of current defined by chemical and/or biochemical reaction taking place within the sensor [2]. Voltamperometric and chemoresistive sensors work similarly in principle but with the difference that the applied voltage is not constant, being changed according to established parameters for a particular method [2, 3].
As a rule amperometric and voltamperometric methods require expensive equipment including an amperometer, external source of voltage or potentiostat, a counter electrode and a reference electrode [2].
Potentiometric devices derive their responses from the change in redox composition of the electroconductive polymer because of the chemical and/or biological reaction, which accompanies the changes in the steady state potential of the potentiometric sensor [2, 4, 5]. The authors of the present invention observe that potentiometric sensors have a number of advantages over amperometric and voltamperometric sensors. One of them is that potentiometric methods do not require sophisticated equipment. A potentiometric device usually comprises a sensor itself, a reference electrode and a high impedance voltmeter [2, 6]. Secondly the signal does not depend on a surface area or a shape of a sensor [2]. Thirdly with a potentiometric method of measurement the problems associated with diffusion processes within the electrochemical cell and resulting in complicated constructions of electrodes (e.g. rotating disc electrodes) for amperometric and voltamperometric methods of measurement, do not play a significant role [2, 4].
The use of potentiometric measurement can be as simple as pH measurement, and the potentiometric device can be similar to commercial pH or ion-selective electrodes.
All electrochemical sensors can be divided into two types, direct and indirect sensors.
A direct electrochemical sensor generates the signal immediately at the moment of interaction between an analyte and receptors immobilised within or adsorbed onto the sensor. Examples of direct sensors are enzyme amperometric sensors [2, 7, 8], ion-selective potentiometric sensors [9, 10] and potentiometric immunosensors [11, 12, 13]. As a rule the contact with an analyte and the measurement procedure are performed simultaneously.
An indirect electrochemical sensor generates a signal due to the presence of additional agents specific to an analyte carrying electrochemically active labels. Examples of the sensors belonging to indirect group are amperometric and potentiometric enzyme sensors [2], potentiometric sensors sensitive to a change in surface potential [14, 15] as well as voltamperometric and chemoresistive sensors [16, 17, 18]. The contact between the sensor and an analyte and the measurement procedure are separated in time and space.
The voltamperometric sensors can be described as intermediate between direct and indirect sensors. In this case there are no labelled agents in the system but the incubation and measurement steps are separated in time and space and/or solute.
The most critical step in the manufacture of a highly sensitive potentiometric sensor, having a conductive polymer layer as a sensing element, is the formation of the polymer film on the conductive support. The support itself is usually a noble metal, carbon or semi-conductive material [19]. Electrochemical synthesis allows production of conductive polymer films with defined chemical, electrochemical and mechanical properties [19].
The components of the polymerisation process include monomer(s), a polar solvent and at least two electrodes (auxiliary and working) [19]. A supporting electrolyte is usually included in the polymerisation solution to increase conductivity of the solution and for doping the polymer at the polymerisation step [19]. There are three main types of electrochemical synthesis: galvanostatic, potentiostatic and potentiodynamic [19].
In the galvanostatic method a constant current from an external source is applied for period of time between working and auxiliary electrodes immersed in the polymerisation solution. The reference electrode may be used to control the electrochemical potential of the working electrode [19, 20, 21].
In the potentiostatic method usually three electrodes are required. The current between the working and auxiliary electrodes is controlled by an amperometer set at a constant voltage from an external source (which is in its turn controlled by the reference electrode) applied between the working electrode and auxiliary electrode for a certain period of time [19, 20, 21].
In the potentiodynamic method the voltage applied between the working and auxiliary electrodes is not constant, but is changing according to established procedures [19, 20, 21].
The most important property for potentiometric enzyme- or immunosensors is their redox sensitivity [4], because most of the enzyme reactions are redox processes accompanied by changing redox state of reactants. The redox sensitivity of polymer-based sensors is completely defined by redox properties of the polymer film [22], which in their turn are defined by the conditions and parameters of the polymerisation process.
A large number of publications are dedicated to research of redox properties of electroconductive polymers, e.g. polypyrrole [5, 22-27]. In a number of these studies [22-23, 26] it was shown that two main mechanisms are employed in the formation of the potentiometric signal. The irreversible change in the intrinsic redox state of the electroconductive polymer is a consequence of the interaction between the polymer layer and electrochemical active species and is referred to as a “corrosive type” of formation potentiometric response. This process is always accompanied by an ionic exchange between the polymer and surrounding solution. Another mechanism is based on an electron exchange between the redox couples on the polymer surface via the polymer film. The intrinsic redox state of the polymer does not change in this process, and an ionic exchange between polymer and solution does not take place. In this case the electroconductive polymer behaves as a metallic potentiometric electrode and its behaviour can be described by the Frumkin theory of electronic equilibrium [22, 28]. This is “metallic type” potentiometric response, and it is reversible. In reality both mechanisms act simultaneously but one of them can be predominant [22-23, 26, 27]. The “metallic type” is favoured for potentiometric redox sensors because it provides the quicker and stronger response [26]. It is possible to change the properties of the polymer film at the polymerisation step making the “metallic” mechanism predominant [22-23, 26].
Previously, the nature of a supporting electrolyte, and accordingly the nature of a dopant ion, were considered the only factors responsible for metallic properties of the polymer film [22-27, 29]. It was shown that the immobile anions embedded within the polymer film do not participate in ion exchange reactions, providing stability of intrinsic redox state [23, 26]. The examples of such electrolytes are dodecylsulphate [23], various dyes, e.g. indigo carmine and methylene blue [30, 31]. However, in all cited publications the concentration of dopant ions in polymerisation solution is not considered as a factor responsible for imparting the metallic properties to the polymer.
The authors of the present invention consider the concentration of the monomer as well as the concentration of supporting electrolyte in the solution for the electrochemical polymerisation as the most important factors for redox properties, and accordingly for redox sensitivity of the polymer. It is known that the concentration of the monomer can also influence the conductivity of polymer [19]. According to the data by the authors of the present invention, the best redox sensitivity can be reached using the polymerisation solution with much lower concentrations of monomer (<0.05M) than commonly used (0.05-0.5M). The authors of the present invention found that the ratio between the concentration of a monomer and supporting electrolyte is a key factor particularly responsible for redox properties and thickness of a polymer film. The ratio between monomer(s) and supporting electrolyte(s) as well as their concentrations had not previously been considered as factors responsible for redox properties of polymer film prior to the studies carried out by the present inventors.
Despite the fact that there are a substantial number of methods for electrochemical synthesis of the electroconductive polymer described in the literature, none of them provide the conditions and parameters for production of highly sensitive sensors suitable for potentiometric detection of biomolecules in low concentration.
Some examples of the prior art methods of electrochemical synthesis are given below.
Potentiostatic methods for preparation of electrochemical polypyrrole-based sensors from aqueous solutions in the presence of a supporting electrolyte are described in [39, 42, 43, 44 and 45].
Taniguchi et al [33] described the method for growing polypyrrole film from organic solvents in the presence of a supporting electrolyte using galvanostatic regime. The generated polymer films were used for the preparation of a direct potentiometric immunosensor. The main disadvantage of this method is that the sensitivity of the sensors produced by this method is poor (mg/ml of IgG). Another disadvantage is that the organic solvents used in such a method are highly toxic.
Other galvanostatic methods where water is used as a solvent are described in [13, 16, 17, 34, 35, 36, 37, 38, 39, 40, 41].
A potentiodynamic method of electrochemical polymerisation for synthesis of electroconductive polymers from aqueous solutions in the presence of a supporting electrolyte for preparation of electrochemical sensors has been described [9, 10, 27, 46, 47, 48, 49].
Most of the sensors produced by the methods described in the articles mentioned above were not intended to use for potentiometric measurement, but for other types of electrochemical measurement or entrapment. All of them are unsuitable for potentiometric assays requiring high analytical sensitivity, precision and stability. The methods cannot be developed directly to the method described in the present invention to provide the required properties of the sensors. The measuring procedures are always more complicated and takes longer than the one which is described in this invention.
The authors of the present invention have also described the potentiodynamic method of preparations of potentiometric polypyrrole-based sensors [50, 51].
Despite the possibility to use polymerisation solutions with low concentrations of monomer(s) in the potentiodynamic and galvanostatic regimes [19], most of the cited publications describe use of concentrations of 0.05M and higher. As it has been stressed by the authors of the present invention, polymer films grown from concentrated monomer solutions do not have high redox sensitivity and therefore cannot be used as a highly sensitive element of the polymer-based potentiometric sensor.
Low concentrations of monomer were mentioned in only one single study [48], but the authors used high concentrations of the supporting electrolytes (0.5M) and supporting electrolytes with highly mobile anions (KCl, KNO3, NaClO4, Na2HPO4), which are actively interactive in ionic exchange between polymer and surrounding solution. The potentiometric response of such sensors belongs mainly to the corrosion-type mechanism. This type of sensor is not sensitive enough for measurement of biological redox reactions, where clinically or environmentally relevant concentrations of analyte may routinely occur in the range of nanomoles, femtomoles or attomoles. Other studies also used highly mobile anions as dopant ions, resulting in sensors, which exhibit low sensitivity. The work of Hulanicki et al [27], where the authors doped polymer with Cl−, can be given as an example. In this case the sensors demonstrated redox sensitivity only in presence of very high concentrations of redox couples (about 0.5M).
Other studies [9, 49, 51] used a suitable dopant ion-sodium dodecylsulphate, but high concentrations of polymer (0.05-0.3M), which is again not suitable for preparation of highly sensitive potentiometric redox sensors.
The present inventors have defined the main factors, which, in combination, are responsible for the redox properties of the polymer film and as a consequence are able to produce polymer-based potentiometric sensors with higher sensitivity than those described in the prior art. These factors are the concentration of a monomer(s); nature and concentration of the supporting electrolyte; parameters of the polymerisation process; ratio between the concentrations of monomer and supporting electrolyte. The prior studies relate to only one or two parameters or conditions and not their synergistic influences or interferences. The authors of the present invention have found that a highly sensitive polymer film can be produced by combining all of the factors mentioned above.